In close collaboration with Philip Anfinrud, novel hardware was designed and developed that demonstrates, for the first time, that it is readily possible to monitor the folding of the protein chain in a residue-specific manner upon jumping the applied pressure. Pressure changes of up to 2.5 kbar, requiring 1-2 ms, are feasible and compatible with the recording of high quality NMR data. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin (VA2-ubiquitin), we have demonstrated that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record two- and three-dimensional NMR spectra of the unfolded protein at atmospheric pressure, providing new, residue specific information on the folding process. Such pressure-jump NMR experiments showed evidence that its folding occurs via two parallel, comparably efficient pathways: A single barrier and a two-barrier pathway. An interrupted folding NMR experiment was developed, where for a brief period the pressure is dropped to atmospheric conditions (1 bar), followed by a jump back to high pressure for signal detection. Conventional, forward sampling of the indirect dimension during the low-pressure period correlates the 15N or 13C' chemical shifts of the unfolded protein at 1 bar to the 1H frequencies of both the unfolded and folded proteins at high pressure. Remarkably, sampling the data of the same experiment in the reverse direction yields the frequencies of structures present at the end of the low-pressure interval, which include unfolded, intermediate, and folded species. Although the folding intermediate 15N shifts differ strongly from natively folded protein, its 13C' chemical shifts, which are more sensitive probes for secondary structure, closely match those of the folded protein and indicate that the folding intermediate must have a structure that is quite similar to the native state. Further experiments to determine an atomic resolution structure of this folding intermediate are in progress. Brain tissue of Alzheimers disease patients invariably contains deposits of insoluble, fibrillar aggregates of peptide fragments of the amyloid precursor protein (APP), typically 40 or 42 residues in length and referred to as Abeta40 and Abeta42. It remains unclear whether these fibrils or oligomers constitute the toxic species. Depending on sample conditions, oligomers can form in a few seconds or less. These oligomers are invisible to solution NMR spectroscopy, but they can be rapidly (< 1 s) resolubilized and converted to their NMR-visible monomeric constituents by raising the hydrostatic pressure to a few kbar. Hence, utilizing pressure-jump NMR, the oligomeric state can be studied at residue-specific resolution by monitoring its signals in the monomeric state. Oligomeric states of Abeta40 were shown to exhibit a high degree of order, reflected by slow longitudinal 15N relaxation (T1 >5 s) for residues 18-21 and 31-34, whereas the N-terminal 10 residues relax much faster (T1 1.5 s), indicative of extensive internal motions. Transverse relaxation rates rapidly increase to ca 1000 s-1 after the oligomerization is initiated, indicating that the oligomers then have accumulated a size on the order of 1 MDa, or ca 250 peptides. Pressure-jump experiments are expected to reveal detailed information on the kinetics of the initiation of oligomer formation, as well as on the details of the structural arrangements of the peptides in the oligomer. Such experiments are currently in progress.